Proteophosphoglycan confers resistance of Leishmania major to midgut digestive enzymes induced by blood feeding in vector sand flies

Leishmania synthesize abundant phosphoglycan‐containing molecules made up of [Gal‐Man‐PO₄] repeating units, including the surface lipophosphoglycan (LPG), and the surface and secreted proteophosphoglycan (PPG). The vector competence of Phlebotomus duboscqi and Lutzomyia longipalpis sand flies was tested using L. major knockout mutants deficient in either total phosphoglycans (lpg2^‐ or lpg5A^‐/5B^‐) or LPG alone (lpg1^‐) along with their respective gene add‐back controls. Our results confirm that LPG, the major cell surface molecule of Leishmania promastigotes known to mediate attachment to the vector midgut, is necessary to prevent the loss of infection during excretion of the blood meal remnants from a natural vector, P. duboscqi, but not an unnatural vector, L. longipalpis. Midgut digestive enzymes induced by blood feeding pose another potential barrier to parasite survival. Our results show that 36–72 h after the infective feed, all parasites developed well except the lpg2^‐ and lpg5A^‐/5B^‐ mutants, which showed significantly reduced survival and growth. Protease inhibitors promoted the early survival and growth of lpg2^‐ in the blood meal. PPG was shown to be the key molecule conferring resistance to midgut digestive enzymes, as it prevented killing of lpg2^‐ promastigotes exposed to midgut lysates prepared from blood‐fed flies. The protection was not associated with inhibition of enzyme activities, but with cell surface acquisition of the PPG, which appears to function similar to mammalian mucins to protect the surface of developing promastigotes against proteolytic damage.     

Expression of the Avirulence gene Avr9 of the fungal tomato pathogen Cladosporium fulvum is regulated by the global nitrogen response factor NRF1

Here we describe the role of the Cladosporium fulvum nitrogen response factor 1 (Nrf1) gene in regulation of the expression of avirulence gene Avr9 and virulence on tomato. The Nrf1 gene, which was isolated by a polymerase chain reaction-based strategy, is predicted to encode a protein of 918 amino acid residues. The protein contains a putative zinc finger DNA-binding domain that shares 98% amino acid identity with the zinc finger of the major nitrogen regulatory proteins AREA and NIT2 of Aspergillus nidulans and Neurospora crassa, respectively. Functional equivalence of Nrf1 to areA was demonstrated by complementation of an A. nidulans areA loss-of-function mutant with Nrf1. Nrf1-deficient transformants of C. fulvum obtained by homologous recombination were unable to utilize nitrate and nitrite as a nitrogen source. In contrast to what was observed in the C. fulvum wild-type, the Avr9 gene was no longer induced under nitrogen-starvation conditions in Nrf1-deficient strains. On susceptible tomato plants, the Nrf1-deficient strains were as virulent as wild-type strains of C. fulvum, although the expression of the Avr9 gene was strongly reduced. In addition, Nrf1-deficient strains were still avirulent on tomato plants containing the functional Cf-9 resistance gene, indicating that in planta, apparently sufficient quantities of stable AVR9 elicitor are produced. Our results suggest that the NRF1 protein is a major regulator of the Avr9 gene.     

Methods for animal cell immobilization using electrostatic droplet generation

Summary A technique for the production of small polymer microbeads (less than 150 m diam.) utilizing electrostatic spraying is presented. Video and image analysis were performed to reveal details of the mechanism of droplet formation. Microbeads coated with poly-L-lysine were prepared and found to be effective as microcarriers for culturing surface immobilized insect cells to a high cell density (1.55 × 10⁷ cells/ml microbeads).     

NMR assignments and secondary structure of the UvrC binding domain of UvrB.

The 55 residue C-terminal domain of UvrB that interacts with UvrC during excision repair in Escherichia coli has been expressed and purified as a (His)6 fusion construct. The fragment forms a stable folded domain in solution. Heteronuclear NMR experiments were used to obtain extensive 15N, 13C and 1H NMR assignments. NOESY and chemical shift data showed that the protein comprises two helices from residues 630 to 648 and from 652 to 670. 15N relaxation data also show that the first 11 and last three residues are unstructured. The effective rotational correlation time within the structured region is not consistent with a monomer. This oligomerisation may be relevant to the mode of dimerisation of UvrB with the homologous domain of UvrC.     

Prolonged survival of transplanted islets of Langerhans encapsulated in a biocompatible membrane

Prolonged survival of islet allografts in streptozotocin-induced diabetic rats was achieved by encapsulating individual islets in protective, biocompatible alginate-polylysine-alginate membranes. A single intraperitoneal transplant of encapsulated islets reversed the diabetic state for up to 1 year. In contrast, a single injection of unencapsulated islets was effective for less than 2 weeks. The microencapsulation procedure, by protecting transplanted tissue from the components of the immune system, has great clinical potential in the treatment of diseases requiring organ transplantation, such as diabetes and liver disease.     

Reorienting in Virtual 3D Environments: Do Adult Humans Use Principal Axes,Medial Axes or Local Geometry?

Studies have shown that animals, including humans, use the geometric properties of environments to orient. It has been proposed that orientation is accomplished primarily by encoding the principal axes (i.e., global geometry) of an environment. However, recent research has shown that animals use local information such as wall length and corner angles as well as local shape parameters (i.e., medial axes) to orient. The goal of the current study was to determine whether adult humans reorient according to global geometry based on principal axes or whether reliance is on local geometry such as wall length and sense information or medial axes. Using a virtual environment task, participants were trained to select a response box located at one of two geometrically identical corners within a featureless rectangular-shaped environment. Participants were subsequently tested in a transformed L-shaped environment that allowed for a dissociation of strategies based on principal axes, medial axes and local geometry. Results showed that participants relied primarily on a medial axes strategy to reorient in the L-shaped test environment. Importantly, the search behaviour of participants could not be explained by a principal axes-based strategy.